Drawing birth

The scid mouse. Characterization and potential uses: Bosma, M.J., Phillips, R.A. & Schuler, W. (Current Topics in microbiology and immunology, 152.) 1 vol. (17 × 24.5 cm), 263 + xii pages. Springer-Verlag, Heidelberg, Berlin, 1989

Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis

Effects of polyunsaturated fatty acids on the proliferation of mitogen stimulated bovine peripheral blood mononuclear cells

Background. The sodium-dependent phosphate transporter NaPi2b is expressed in a number of tumors and serves as a target for monoclonal antibody therapy. The NaPi2b transporter has a large extracellular domain containing 4 cysteines and an MX35 epitope recognized by the corresponding monoclonal antibodies. Aim. To study the recognition of the MX35 epitope in cysteine mutants C303 and C350 of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian cancer cells. Material and methods. Ovarian cancer cells of the OVCAR-8 line were transfected with plasmids encoding mutant forms of the NaPi2b transporter. The recognition of these forms by monoclonal antibodies was studied by Western blot and flow cytometry. Statistical analysis of flow cytometry data was performed using Pearsons Chi-squared test with Yates correction. Results. Western blot analysis of lysates of OVCAR-8 cells expressing the wild-type NaPi2b transporter demonstrated the presence of a specific signal only in samples without the addition of dithiothreitol, while in all samples with NaPi2b cysteine mutants, even without the addition of dithiothreitol, no specific signal from monoclonal antibodies was detected. The flow cytometry results showed that proportion of OVCAR-8 cells with positive staining of the MX35 epitope with antibodies is less when expressing the NaPi2b mutants compared to the wild type. Conclusion. Cysteines C303 and C350 are involved in disulfide bonds formation, which is of key importance for the formation of the conformation of the extracellular domain of the sodium-dependent phosphate transporter NaPi2b, which ensures the MX35 epitope recognition by monoclonal antibodies.

Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells: Functional evaluation of monocytes in responses to lectins

In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status

Bovine herpesvirus 1 (BHV-1) is able to inhibit the proliferation of bovine peripheral blood mononuclear cells. Here, we have demonstrated that live BHV-1 and, interestingly, inactivated BHV-1 can induce apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.

The effects of cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) on the cellular metabolic activity and activation status of bovine peripheral blood mononuclear cells (PBMC) were investigated. Cellular DNA and protein synthesis was determined by [3H]thymidine and [3H]valine incorporation, respectively, in phytohemagglutinin (PHA)-stimulated PBMC. All cp strains and most ncp BVDV strains significantly inhibited DNA synthesis in PHA-stimulated PBMC; however, only cp BVDV strains inhibited protein synthesis. A plaque assay and immunofluorescence test confirmed productive BVDV infection of PBMC. In addition, viral RNA synthesis was demonstrated in BVDV-infected PBMC by RT-PCR. The interleukin-2 receptor (IL-2R) was used as a marker for the activation status of BVDV-infected PBMC. The expression of IL-2R was preserved in virus-infected cells, even though DNA and protein synthesis was suppressed. These findings suggest a novel mechanism of virus-induced immune suppression in which BVDV inhibits basic metabolic activities of bovine PBMC. The activation signals, however, are maintained.

Death receptor 3 (DR3) is a tumor necrosis factor receptor superfamily member (TNFRSF25), which is minimally expressed on resting conventional T cells (though readily inducible upon cell activation), yet highly expressed on resting FoxP3+ regulatory T cells (Treg). We recently demonstrated that activation of DR3 with an agonistic antibody (4C12) leads to selective expansion and activation of Treg in healthy mice and suppression of graft-versus-host disease (GVHD) in recipient mice when donor mice are treated. However, given the long antibody half-life and concomitant safety concerns, along with the lack of a humanized agonistic antibody to DR3, both human and murine fusion proteins incorporating the natural DR3 ligand TL1A (TL1A-Ig) have been developed. Herein, we show that DR3 activation with 4C12 or with TL1A-Ig, with or without the addition of low dose IL-2 to the treatment regimen, led to a significant expansion of murine Treg in spleen, lymph nodes, and peripheral blood. Bioluminescent imaging revealed peak Treg expansion around day 7–8, with return to near baseline after 2–3 weeks. In addition to expansion, all DR3 agonist treatment regimens led to increased activation of Tregs, with significant upregulation of the activation markers ICOS, KLRG-1, PD-1, and CD103, and the proliferation marker Ki-67. The near absence of activated Treg populations in control treated spleens was also detected on tSNE analysis of flow cytometry data. Subtly different patterns of splenic Treg activation by the different DR3 agonists were noted in both tSNE analysis of flow cytometry data and RNA-sequencing analysis. However, upregulation of gene transcripts which play important roles in cell proliferation, trafficking, activation, and effector function were observed regardless of the DR3 agonist treatment regimen used. In the major MHC-mismatch model of hematopoietic cell transplantation, DR3 agonist-mediated expansion and activation of Tregs in donor mice led to a significant improvement in GVHD in recipient mice. These data provide important preclinical information regarding the outcome of DR3 activation with an agonistic antibody or natural ligand and provide insight into the therapeutic use of this approach to reduce GVHD in recipients and improve outcomes of hematopoietic cell transplantation.

Phenotypic and functional characteristics of bovine T lymphocytes

Effects of recombinant human cytokines on mitogen-induced bovine peripheral blood mononuclear cell proliferation

Enterovirus eibovi (EV-E) is a bovine RNA virus with debated pathogenic potential. The aim of this study was to evaluate the in vitro effects of bovine lactoferrin (bLF), an antimicrobial and immunomodulatory glycoprotein, on EV-E-infected bovine peripheral blood mononuclear cells (PBMC). The PBMC viability, phagocytic function, and lymphocyte proliferation were examined using spectrophotometric assays. Flow cytometry was used for immunophenotyping of lymphocytes, and real-time quantitative PCR for evaluation of immune-related gene expression. The safe concentration range of bLF (1-25 µg/mL) was determined on the basis of the viability and proliferation of uninfected PBMC. Bovine lactoferrin significantly influences the activity of enterovirus-infected cells, with high (10 and 25 µg/mL) and low (1, 2.5, and 5 µg/mL) protein concentrations exerting different effects. Low concentrations of bLF primarily affected unstimulated cells, increasing the expression of IFNα, IFNβ, IL2, and NFκB genes. In contrast, high concentrations of lactoferrin predominantly modulated the activity of infected cells that were additionally stimulated with a mitogen or antigen: They increased T lymphocyte proliferation; reduced phagocyte activity; increased the expression of IFNα, IL10, and NFκB; and decreased the expression of IFNγ. The increased expression of IL10 in response to high protein concentrations was also observed in unstimulated cells. The only common effect of both high and low concentrations of bLF was a decrease in the percentage of WC1+ γδ T cells following concanavalin A stimulation. Thus, bLF modulated the immune response of EV-E-infected PBMC in a concentration-dependent manner: Lower concentrations promoted early antiviral responses by inducing type I interferons and IL-2, whereas higher concentrations upregulated the anti-inflammatory cytokine IL-10, suggesting a protective effect against immune overstimulation. These findings indicate that bLF may serve as an immunomodulatory agent during enteroviral infections in cattle, with promising implications for both therapeutic and prophylactic applications.

Flow cytometry is a technique for analyzing cells that are suspended in a buffered salt-based solution and flow past one or more lasers. Visible light scatter and one or more fluorescence characteristics are assessed for each particle. The most common use of flow cytometry is immunophenotyping. In traditional flow cytometry analysis, a region around a population of cells is manually drawn (gating) in two-dimensional scatter plots. This enables the measuring of specific groups of cells. However, manual gating could increase the overall error rate of the study and makes the analysis hardly reproducible since it is done in less controlled settings. Furthermore, it represents a bottleneck in the analysis of large amounts of data. Some processes, such as gating, have recently been automated using R packages. In addition, dimensionality reduction approaches have been developed in the flow cytometry environment to take advantage of information from several markers at once. Despite this, none of them are integrated in a harmonized way, and none of them allow back-gating to emphasize the study population and eliminate false positives. To tackle this problem, our team has developed flowTOTAL (github.com/ImmunoOncology/flowTOTAL), a user-friendly command line workflow to analyze flow cytometry data. The major attractive feature is the facility to perform with one command not only a traditional analysis, but also an unsupervised analysis. As input, the user has to indicate the folder with the .FCS files, the metadata associated with each file, and the marker to be used during back-gating. The pipeline is divided into three main sections: preprocessing, traditional analysis, and unsupervised analysis. During preprocessing each. FCS will be subjected to correcting for fluorescence spillover (compensation), detection of anomalies by checking flow rate and signal acquisition as well as removing doublets based on forward scatter (QC). For the traditional analysis, auto-gating will be performed for the identification of the target population using back-gating. For each set of given markers, the number of events obtained and the scatter plot will be generated. Finally, in the unsupervised analysis, the population of interest will be specified and the pipeline will proceed with normalization, dimensionality reduction using PCA or UMAP and finally a clustering approach for subpopulation identification. In addition, differential abundance analysis can be performed with metadata information. flowTOTAL is presented as a standardization for the analysis of flow cytometry data, comprising all the necessary steps for comprehensive analysis and allowing mass analysis. Furthermore, it goes beyond the simple quantification of particles, since the implementation of more complex methodologies allows for the discovery of subpopulations that are not present in the traditional analysis but have a significant biological role. Citation Format: Juan Luis Onieva, Patricia Cháves, Javier Oliver, María Garrido-Barros, Juan Zafra, Belén Sojo, Alfonso Sánchez, Martina Álvarez, Pedro Jiménez, Emilio Alba, Miguel Berciano, Antonio Rueda, Manuel Cobo-Dols, Elisabeth Pérez, Isabel Barragán. flowTOTAL: A comprehensive bioinformatics workflow for flow cytometry automatic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1910.

Expression of the bovine high affinity IL-12 receptor β2

Monoclonal antibody specific for bovine CD 5 antigen which enhances mitogen-induced blastogenesis and IL-2 production